Troubleshooting Western Blot Experiments

Although western blot workflows have changed since the technique was first developed, the fundamental principles have remained the same. Consequently, the types of problems faced by researchers have stuck around. Here, we comment on how western blot has evolved, before suggesting ways of addressing common issues.

Western blot improvements

For over 40 years, western blot has retained its status as one of the most widely used protein detection methods in life science research. But modern western blotting is much improved compared to early forms of the technique. “Improvements to western blot across reagents, instrumentation, and software have led to better reproducibility, time to results, and throughput,” reports Emily Halbrader, Sr. Manager, Product Management, Biosciences Division at Thermo Fisher Scientific. “For example, innovative precast gel chemistries such as Bis-Tris Bolt provide neutral pH conditions and can be run at faster times with increased sample load volumes for better protein resolution and detection analysis. Newly developed high sensitivity chemiluminescent substrates enable detection down to the attogram level to help with identifying low abundance targets. And modern gel documentation and western blot imaging systems can provide faster image capture, higher resolution, and more accurate data analysis.”

According to Joy De Torres, Applications Scientist at Azure Biosystems, advancements in digital imaging technology have been the biggest factor in changing the western blot landscape to date. “Modern digital imaging has increased the sensitivity and dynamic range for western blot detection, moving us from the qualitative assay that western blots used to be toward the quantitative assay that western blots can be today,” she notes. “Imaging systems are now available that promote following western blot best practices—such as normalization against a total protein stain rather than a housekeeping protein—meaning data is far more likely to meet the increasingly rigorous standards required for publication.”

For an in-depth overview of emerging techniques for western blot, Cali Anderson, Technical Specialist at Proteintech Group, recommends a recent Future Journal of Pharmaceutical Sciences paper. This includes a comparison of different transfer methods whose use, like any stage of the western blot workflow, should always be considered with respect to the target being investigated. “While the authors describe great improvements in transfer speed and efficiency, it is worth noting that a quick high-voltage transfer is not always the answer, especially for larger proteins,” says Anderson. “Taking the time to carefully plan each step of your western blot will help prevent wasted resource and sample material as experiments are repeated.”

Troubleshooting

Western blot problems generally fall into one of several main categories, of which weak signal, unexpected bands, and uniform high background are among the most common. We’ve suggested ways of addressing these issues here, and you can also find further help in our Western Blot Troubleshooting Guide.

Weak or no signal

“Weak or no signal is most often seen when antibody dilution and substrate conditions are not fully optimized, and can be further exacerbated if sub-optimal exposure times are used during imaging,” comments Halbrader. “To avoid this, it is best to start with the manufacturer’s recommended dilutions for the primary antibody and follow the secondary antibody dilution guidelines in conjunction with a high-quality substrate. Additionally, leveraging an auto-exposure feature during imaging can help to improve the signal-to-noise ratio without over- or under-exposing, thereby avoiding saturated or weak signal.”

Incorrect antibody storage can also be responsible for weak or no signal. “It is important to be mindful of the storage buffer and to view the recommended storage conditions from the antibody manufacturer, as they will have determined how best to ensure antibody longevity and accurate results,” says Anderson. “For example, some antibody storage buffers contain 50% glycerol to protect against freeze-thaw, so there is no need for aliquoting. This circumvents the problem of antibodies sticking to the plastic PCR tubes commonly used for storing aliquots, which decreases the available antibody concentration.”

Other possible reasons for weak or no signal include poor transfer, which can easily be checked using Ponceau-S or a total protein stain, and incorrect membrane selection. Sometimes, simply switching from nitrocellulose to PVDF can dramatically increase the target signal due to the different mechanisms involved in protein binding.

Multiple or wrong size bands

“The presence of multiple bands, or bands that do not match the expected molecular weight of the target protein, can often be attributed to poor antibody specificity,” explains De Torres. “However, it may also be the case that your antibody recognizes several different protein isoforms or a modified form of the target. My advice here is to confirm that your chosen antibody is validated for western blot and to refer to sites such as uniprot.org to learn all you can about your protein of interest. You can then try implementing strategies such as performing the primary antibody incubation at 4^oC and optimizing the blocking conditions to reduce non-specific binding.”

‘Too many bands’ can frequently be attributed to problems with lysate preparation. “Older lysates can have significant protease activity and are more prone to spontaneous degradation,” explains Anderson. “For this reason, you should use fresh lysates where possible, always including protease inhibitors in the lysis buffer and performing the lysate prep on ice.” Samples should also be boiled immediately after extraction so that any proteases in the samples lose activity. However, for heat-sensitive membrane proteins, this step should be avoided since it can lead to the formation of insoluble aggregates.

A less obvious reason for seeing multiple bands on a western blot is not loading enough protein. Anderson clarifies that while this might seem counterintuitive, non-specific antibody binding is energetically favored at lower protein concentrations, so increasing the amount of protein loaded can have drastic effects in detecting the correct band. Critically, the potential significance of unexpected bands should never be ignored, since this could lead to key information being overlooked.

Uniform high background

Uniform high background on a western blot can obscure bands of interest. Ways of addressing this include switching to a different blocking buffer, titrating antibody reagents, and using more stringent washing conditions. “Fluorescent blots can especially benefit from washing with higher detergent concentrations followed by a rinse in PBS to reduce unwanted background,” comments De Torres. In addition, when using high sensitivity chemiluminescent substrates, adhering to the manufacturer’s recommended secondary antibody concentration is essential. “This can often be up to tenfold lower than that required for standard ECL detection,” cautions Anderson. “Not using the suggested concentration could therefore lead to fully black blots!”

Manufacturers are here to help

While the problems just discussed are some of the most prevalent, they are far from being the only issues encountered when running western blots. Smeared lanes, ‘smiling’ bands, and inconsistent data between experiments are other frequent complaints, along with issues that are unique to different detection methods. Fortunately, manufacturers of western blotting reagents and instrumentation will probably have seen these before and can offer expert technical support, often on an application-specific basis. “Using high-quality, optimized product solutions that are tailored for western blotting is an essential first step toward generating reliable data,” notes Halbrader. “Anyone can get great western blot results with the right tools and support, so speak with manufacturers to help identify the most appropriate products for your experiment.”